Although knowledge about molecular mechanism of cell differentiation and tissue formation is accumulating very quickly, it is still unclear how the size of an organ is regulated. 40 years ago, Bullough proposed a hypothesis that a negative growth regulator called the “chalone (an endocrine substance inhibits the physiological action)” regulates the size of an organ [Bullough W S., Cancer Res., 25, 1683, 1965]. According to the hypothesis, when an organ grows to a certain size, it secretes the chalone so that it cannot grow further. The chalone was not discovered in spite of extensive researches over the last 40 years, and the hypothesis seemed to be abandoned. But, recently, a chalone called myostatin was found in skeletal muscle.
Myostatin (myo means muscle, and statin means stop), a member of the transforming growth factor-β superfamily, is expressed in skeletal muscle. A significantly increased skeletal muscle mass was found in myostatin-deficient mouse [McPherron A C. et al., Nature, 387, 83, 1997]. Myostatin mutation was found in not only in double-muscled Belgian Blue and Pidemontese cattle [McPherron A C. et al., PNAS, 94, 12457, 1997], but also in with gross muscle hypertrophy [Schuelke, M. et al., New Eng. J. Med., 350, 2682, 2004]. These findings prove that myostatin is the chalone that regulates the size of muscle. In myostatin −/− mice, the muscle mass increases while the accumulation of fats decreases remarkably. When obese (ob/ob) mice were bred with myostatin −/− mice, the offspring exhibited normal body weight, reduced fats and normal blood sugar level [McPherron A C. and Lee, S J., J. Clin. Invest., 109, 595, 2002]. This indicates that obesity and type 2 diabetes may be treated by reducing the amount of myostatin or inhibiting its activity. In addition, it was shown that when mice with muscular dystrophy induced by dystrophin deficiency were treated with anti-myostatin antibody, muscle length and diameter were restored as those of normal mice [Bongdanovich S., Nature, 420, 416, 2002]. This implies that anti-myostatin antibody may be used for the treatment of muscular dystrophy patients. Actually, Wyeth (USA) is carrying out clinical trials on myostatin. However, because myostatin-deficient mice and myostatin mutant cattle lose their reproductive ability [Animal Genetics, 36, 1, 2004; J. Gene Med., 8, 1171, 2006, drugs using myostatin as a target may result in severe adverse reactions.
Whereas myostatin regulates muscle size negatively, insulin-like growth factor (IGF-I) regulates it positively. For example, mice in which IGF-I was overexpressed only in muscle using muscle-specific promoter exhibited two times more muscle mass than normal mice [Masaro A. et al., Nature Genet., 28, 195, 2001]. Thus, there was a medical attempt to use IGF-I as muscle enhancer. But, this attempt met serious obstacles. For one thing, because IGF receptors are expressed in nearly every cell, the administration of IGF-I may have a serious effect not only on muscle but also on other tissues. Besides, IGF-I promotes cell division and proliferation, thereby resulting in unwanted hyperproliferation of tissues. Accordingly, it is needed to induce selective expression of IGF-I only in muscle (through genetic treatment) or selective IGF-I signaling only in muscle.
FIG. 1 schematically illustrates the IGF-I signaling. When the signaling pathway IRS-1→PI3K→AKT-1→mTOR→p70S6K which is activated by IGF-I is blocked (using mice with deficiency of each gene), the mice become smaller in size and the muscle mass decreases abruptly. And, in PTEN- and SHIP2-deficient mice, the amount of Ptd Ins(3,4,5)P3 increases to induce overexpression of PI3K/AKT1. As a result, the muscle mass increases [Glass, D J., Nat. Cell Biol., 5, 87, 2003]. These results show that PI3K, AKT-1, mTOR, p70S6K and GSK3β might be good candidate target proteins for the development of muscle enhancer. However, because these proteins affect not only muscle but also other tissues, activation (PI3K, AKT-1, mTOR, p70S6K or GSK3β) or inhibition (PTEN or SHIP2) using the proteins may lead to adverse reactions such as cancer. Accordingly, these proteins are not good targets for the development of muscle enhancer.
The inventors of the present invention isolated lipid rafts from C2C12 myoblasts and myotubes, and identified TRIM72, the function of which was never known yet, through comparative proteomics. TRIM72 has a (ubiquitin E3) TRIM/RBCC domain necessary for ubiquitin E3 ligase activity. TRIM72 is specifically expressed in lipid rafts of skeletal muscle and heart and results in increased muscle mass during muscle differentiation. Overexpression of TRIM72 inhibits myogenesis, whereas knockdown of TRIM72 enhances myogenesis. This signifies that TRIM72 is a negative regulator of skeletal muscle differentiation. TRIM72 regulates the IGF-I/IGFR/IRS-1 signaling pathway by inhibiting IGF-1-mediated activation of IRS-1 through interaction with IRS-1. Accordingly, TRIM72 acts only on skeletal muscle and heart, without affecting the IGF-I signaling pathway in other tissue. Therefore, cancer or other side reactions may be prevented. That is, a drug or genetic treatment using TRIM72 as target may lead to the treatment of obesity and type 2 diabetes without side effects, by promoting differentiation and hypertrophy of skeletal muscle and energy consumption in adipose tissue, and to a strong heart, by promoting physiological hypertrophy of heart muscle.
[Disclosure]
[Technical Problem]
In an aspect, there is provided a new use of TRIM72 as a target for muscle and heart enhancer.
In another aspect, there is provided a composition for enhancing muscle or heart comprising an expression or action inhibitor of the TRIM72 protein.
In another aspect, there is provided a method for enhancing muscle or heart, which comprises administering an expression or action inhibitor of the TRIM72 protein.
In another aspect, there is provided a new TRIM mutant protein or its gene having activity of promoting muscle differentiation and hypertrophy.
[Technical Solution]
According to an embodiment of the present invention, there is provided a composition for enhancing muscle or heart comprising an expression or action inhibitor of TRIM72 protein as an effective ingredient. It is first proposed in the present invention that inhibition of expression or action of TRIM72, a negative regulator of muscle differentiation, promotes muscle differentiation and hypertrophy and may lead to enhancement of muscle or heart.
TRIM72 is an abbreviation of tripartite motif-containing 72 and has a RING domain, a B box, a coiled-coil domain and a SPRY domain, as shown in FIG. 3C. Base sequence and amino acid sequence of mouse TRIM72 may be obtained from NCBI Accession No. NM—001079932. The amino acid sequence of mouse TRIM72 protein is given as SEQ ID NO: 1. Base sequence and amino acid sequence of human TRIM72 may be obtained from NCBI Accession No. NM—001008274. The amino acid sequence of human TRIM72 protein is given as SEQ ID NO: 2. The mouse and human TRIM72 are expected to have the same function, because their amino acid sequences have 90% identity. But, their function was not elucidated as yet.
For the expression or action inhibitor of TRIM72 protein, any substance known to inhibit expression or action of protein may be used. Preferably, a composition for enhancing muscle or heart which downregulates transcription or translation of TRIM72 gene or inhibits action of TRIM72 protein is provided. As used herein, “downregulation of transcription or translation of TRIM72 gene” includes downregulation of transcription by binding to the TRIM72 promoter gene, degradation of mRNA after transcription, interruption of translation, or any other downregulation. And, “inhibition of action of TRIM72 protein” includes inhibition of protein activity and interruption of protein interaction with other proteins by competitively binding.
Examples of the expression inhibitor of TRIM72 protein may include siRNA (short interfering RNA) using RNA interference of TRIM72 gene or shRNA (short hairpin RNA). RNA interference (hereinafter, “RNAi”) is a mechanism that inhibits gene expression after transcription in many eukaryotes. RNAi is induced by short double-stranded RNA (“dsRNA”) molecules existing in cells [Fire A. et al., (1998), Nature 391: 806-811]. These short dsRNA molecules also known as the “siRNA” are separated into single strands and bind to “RNA-induced silencing complex (RISC)”, thereby cleaving target mRNA or interfering translation [Elbashir S M et al., (2001), Genes Dev., 15: 188-200].
Thus, the present invention provides a separated siRNA comprising short double-stranded RNA consisting of from about 17 to about 25 nucleotides targeting mRNA of TRIM72 gene. The siRNA comprises a sense RNA strand and its complementary antisense RNA strand. These two strands bind (anneal) with each other through Watson-Crick base pairing interaction. The sense strand includes the same nucleotide sequence in the target sequence of the target mRNA. The target sequence of siRNA may be selected by a method published in the literature, e.g., [Tuschl T. et al., “The siRNA User Guide” revised Oct. 11, 2002]. The TRIM72 target sequence used to manufacture the siRNA of the present invention is 5′-AAGCACGCCUCAAGACACAG-3′.
The sense and antisense strands of the siRNA of the present invention may include two complementary, single-stranded RNA molecules, or a molecule wherein two complementary moieties are base-paired and covalently bonded by a single-stranded “hairpin” domain. The latter is called shRNA (short hairpin RNA). shRNA is a single strand, about 50-70 nucleotides in length, having a stem-loop structure in vivo. On both sides of 5-10 nucleotide loop portion, long RNA of 19-29 nucleotides are base-paired to form a double-stranded stem. In general, shRNA is synthesized in vivo from the Pol III promoter by the transcription of complementary DNA sequence. The Pol-III-induced transcription starts from the well-defined start site and terminates at the linear second residue consisting of 4 or more thymidines (-TTTT-) to form a non-poly(A) transcript. The Pol III promoter is activated in all cells and can express the shRNA. Following the transcription, the shRNA has its loop cleaved by Dicer, and interacts with RISC like siRNA [see Tuschl, T. (2002), Cell 110(5): 563-74].
The siRNA of the present invention may be obtained by the method well known to those skilled in the related art. For example, the siRNA may be synthesized chemically or produced by recombinant technique using the method well known in the related art. Preferably, the siRNA of the present invention may be synthesized chemically using adequately protected ribonucleoside phosphoramidites and a commonly used DNA/RNA synthesizer. The siRNA may be synthesized as two separated complementary RNA molecules or as an RNA molecule having two complementary domains. Alternatively, the siRNA may be expressed from a recombinant DNA plasmid using an adequate promoter. Examples of the adequate promoter for expressing the siRNA of the present invention from plasmid may include U6 or H1 RNA pol III promoter and cytomegalovirus promoter. Further, the recombinant plasmid may include an inducing promoter or a controllable promoter so that the siRNA can be expressed under a specific tissue or cell environment.
The siRNA of the present invention may be expressed from the recombinant plasmid as two separated complementary RNA molecules or as an RNA molecule having two complementary domains. Selection of adequate plasmid for expressing the siRNA of the present invention, insertion of nucleotide sequence for expressing the siRNA into the plasmid, and transfer of the recombinant plasmid to target cells are disclosed in the related art. For example, refer to the literatures [Tuschl, T. (2002), Nat. Biotechnol., 20: 446-448; Brummelkamp T R et al. (2002), Science 296: 550-553; Miyagishi M. et al., (2002), Nat. Biotechnol. 20: 497-500; Paddison P J et al. (2002), Genes Dev. 16: 948-958; Lee N S et al. (2002), Nat. Biotechnol. 20: 500-505; and Paul C P et al. (2002), Nat. Biotechnol. 20: 505-508], which are incorporated herein by reference.
In the composition for enhancing muscle or heart of the present invention, the expression inhibitor of TRIM72 protein may be TRIM72 siRNA (short interfering RNA) having a base sequence complementary to mRNA of TMRIM72 gene, more preferably a base sequence of SEQ ID NO: 3, or a gene which transcribes the same.
In the composition for enhancing muscle or heart of the present invention, the action inhibitor of TRIM72 protein may be a substance that interferes with the interaction of TRIM72 with IRS-1 or M-Cadherin. As it was found out that the coiled-coil domain of TRIM72 binds with the Mid and Rear regions of IRS-1 (see FIG. 9), a peptide which binds specifically to the binding domain and can competitively inhibit the interaction may be designed. Further, polyclonal or monoclonal antibody against TRIM72 may be used as the interaction inhibitor.
The polyclonal antibody may be prepared by injecting the immunogen TRIM72 protein or its fragment into a host by the method well known to those skilled in the art. The host may include a mammal such as mouse, rat, sheep and rabbit. The immunogen may be injected intramuscularly, intra-abdominally or subcutaneously. An adjuvant may be administered together in order to enhance antigenicity. Blood is taken from the host at predetermined intervals to collect the serum showing improved titer or antigen specificity, or antibody is isolated and purified therefrom.
The monoclonal antibody may be prepared by fusing with an immortalized cell line as well known in the related art [Koehler and Milstein (1975) Nature, 256:495]. The procedure is as follows. First, 20 μg of pure TRIM72 protein is obtained and Balb/C mouse is immunized using the same, or a peptide is synthesized and bound to bovine serum albumin and the mouse is immunized using the same. Then, antigen-producing lymphocyte isolated from the mouse is fused with human or mouse myeloma to produce immortalized hybridoma, or only the hybridoma cells producing monoclonal antibody are selected and proliferated by ELISA, and monoclonal antibody is isolated and purified from therefrom.
In the composition for enhancing muscle or heart of the present invention, the expression or action inhibitor of TRIM72 protein may enhance IGF-1-induced PI3K/AKT activation. The function of the inhibitor is demonstrated in Result 4.
The present invention further provides a composition for inhibiting muscle differentiation wherein tyrosine phosphorylation of IRS-1 is enhanced by the expression or action inhibitor of TRIM72 protein so as to improve interaction between IRS-1 and PI3K. The function of the inhibitor is demonstrated in Result 5.
In the composition for inhibiting muscle differentiation of the present invention, the expression or action inhibitor of TRIM72 protein may inhibit ubiquitination of IRS-1 or M-Cadherin induced by TRIM 72. The function of the inhibitor is demonstrated in Results 7 and 8.
In another embodiment, the present invention provides a method for screening muscle or heart enhancer comprising: (a) treating an agent in animal cells expressing TRIM72 protein; and (b) determining whether the expression of TRIM72 protein decreases as compared to a control group in which the animal cells are not treated with the agent. In the step (a), the animal cells may be intentionally transfected with a plasmid overexpressing TRIM72. In the step (b), the decrease of expression may be measured by RT-PCR in RNA level, or by Western blotting, etc. in protein level.
In another embodiment, the present invention provides a method for screening muscle or heart enhancer comprising: (a) treating an agent in animal cells expressing TRIM72 protein; and (b) determining whether the interaction of TRIM72 with IRS-1 or M-Cadherin is inhibited in the animal cells. In the step (a), the animal cells may be intentionally transfected with a plasmid overexpressing TRIM72. In the step (b), the inhibition of interaction may be measured by immunoprecipitation, GST pull-down assay, or the like.
In another embodiment, the present invention provides a composition for inhibiting muscle differentiation comprising a TRIM72 protein having an amino acid sequence of SEQ ID NO: 1 or 2 or a gene encoding for the same. It is first elucidated in the present invention that TRIM72 is a negative regulator of muscle differentiation and proposed that it can inhibit muscle differentiation by blocking IGF-1 signaling necessary for the muscle differentiation.
In the composition for inhibiting muscle differentiation of the present invention, the gene may be inserted in an animal cell expressing vector, more preferably in an adenoviral vector. For the plasmid vector, pCMV-Flag or pHM6-HA vector may be used. And, for the adenoviral vector, pAd/CMV/V5-DEST (Invitrogen) may be used.
In the composition for inhibiting muscle differentiation of the present invention, the TRIM72 protein may inhibit IGF-1-induced PI3K/AKT activation. The function of the TRIM72 protein is demonstrated in Result 4.
In the composition for inhibiting muscle differentiation of the present invention, the TRIM72 protein may bind to IRS-1 and reduce tyrosine phosphorylation of IRS-1, thereby inhibiting the interaction of IRS-1 with PI3K. The function of the TRIM72 protein is demonstrated in Result 5.
In the composition for inhibiting muscle differentiation of the present invention, the TRIM72 protein may promote ubiquitination of IRS-1 or M-Cadherin by interacting with them. The function of the TRIM72 protein is demonstrated in Results 7 and 8.
In another embodiment, the present invention provides an siRNA (short interfering RNA) molecule for TRIM72 gene having a base sequence of SEQ ID NO: 3. The siRNA targets 5′-AAGCACGCCUCAAGACACAG-3′ of TRIM72 mRNA and has a sequence complementary to it.
In another embodiment, the present invention provides a TRIM72 mutant protein (ΔRING TRIM72) in which the RING domain is deleted from an amino acid sequence of SEQ ID NO: 1 or 2. SEQ ID NO: 1 is the amino acid sequence of mouse TRIM72. Of its RING domain (1st through 60th amino acids), all or part may be deleted. SEQ ID NO: 2 is the amino acid sequence of human TRIM72. Of its RING domain (1st through 60th amino acids), all or part may be deleted.
The present invention further provides a TRIM72 mutant protein having an amino acid sequence of SEQ ID NO: 4 or 5. SEQ ID NO: 4 is the amino acid sequence of mouse ΔRING TRIM72, and SEQ ID NO: 5 is the amino acid sequence of human ΔRING TRIM72.
In another embodiment, the present invention provides a TRIM72 mutant gene which has a base sequence encoding for the TRIM72 mutant protein (ΔRING TRIM72). The base sequence may be easily designed by deleting only the RING domain coding sequence from the base sequence of mouse or human TRIM 72 gene.
In another embodiment, the present invention provides a TRIM72 mutant protein (DN-TRIM72) in which the 14th amino acid in an amino acid sequence of SEQ ID NO: 1 or 2 is substituted from cystine to alanine.
In another embodiment, the present invention provides a TRIM72 mutant gene which has a base sequence encoding for the RIM72 mutant protein (DN-TRIM72). The base sequence may be easily designed by substituting the 14th codon in a base sequence of a mouse or human TRIM 72 gene from a cystine codon to an alanine codon.
In another embodiment, the present invention provides a composition for enhancing muscle or heart comprising the TRIM72 mutant protein or the gene encoding for the same. It is first elucidated in the present invention that the TRIM72 mutant is a dominant negative form of TRIM72 and proposed that it can enhance muscle differentiation and hypertrophy, thereby strengthening muscle or heart.
In the composition for enhancing muscle or heart of the present invention, the gene may be inserted in an animal cell expressing vector, more preferably an adenoviral vector. For the plasmid vector, pCMV-Flag or pHM6-HA vector may be used. And, for the adenoviral vector, pAd/CMV/V5-DEST (Invitrogen) may be used.
In another embodiment, the present invention provides an animal cell expressing vector comprising the TRIM72 mutant gene.
The animal cell expressing vector may be an adenoviral vector.
In another embodiment, the present invention provides a virus clone obtained by packaging an adenoviral vector comprising the TRIM72 mutant gene in 293T cells.
In another embodiment, the present invention provides animal cells transformed by the animal cell expressing vector comprising the TRIM72 mutant gene.
In another embodiment, the present invention provides a TRIM72 promoter in which the E2 or E3 box is truncated or mutated from a base sequence of any of SEQ ID NOS: 6 to 8. The base sequences of SEQ ID NOS: 6 to 8 are those of TRIM72 promoters comprising the domains 1269 bp, 1144 by and 883 bp, respectively, upstream of the ATG start codon. The mutant TRIM72 promoter of the present invention may be one in which the E2 box, which corresponds to 557th through 562nd upstream of the start codon, or the E3 box, which corresponds to 580th through 585th upstream of the start codon, is truncated or mutated.
The TRIM72 promoter may have a base sequence of SEQ ID NO: 9 or 10. SEQ ID NO: 9 is that of an 883 by TRIM72 promoter in which the E2 box is mutated, and SEQ ID NO: 10 is that of an 883 by TRIM72 promoter in which the E3 box is mutated.
Because the TRIM72 proposed by the inventors negatively controls the growth of muscle, cattle with large muscle mass may be produced by knocking out the TRIM72 gene. Also, cattle with large muscle mass may be produced by inducing transgenic cattle overexpressing the mutant ΔRING TRIM72 or C14A TRIM72.
The pharmaceutical composition of the present invention may be administered along with a physiologically acceptable vehicle. For oral administration, a binder, glidant, disintegrant, excipient, solubilizer, dispersant, stabilizer, suspender, pigment, perfume, etc. may be used. For injection, a buffer, preservative, pain reliever, solubilizer, tonicity agent, stabilizer, etc. may used. For local administration, a base, excipient, preservative, etc. may used.
The pharmaceutical composition of the present invention may be prepared into various formulations along with the aforementioned pharmaceutically acceptable vehicles. To take oral administration for example, it may be prepared into tablet, troche, capsule, elixir, suspension, syrup, wafer, or the like. For injection, it may be prepared into a single administration ampule or multiple administration form. Besides, the pharmaceutical composition of the present invention may be prepared into various formulations according to the method commonly used in the related art.
The pharmaceutical composition of the present invention may be orally or parenterally, e.g., intravenously, subcutaneously, intranasally or intra-abdominally, to human and animals. Parenteral administration may be carried out by injection, e.g., subcutaneous injection, intramuscular injection and intravenous injection, or dropping.
The effective dose of the TRIM72 gene, TRIM72 protein or its inhibitor included in the pharmaceutical composition of the present invention may vary dependent on sex, severity of disease, age, route of administration, target cells, degree of expression, and the like, and may be easily determined by those skilled in the art. For example, the effective dose may be from about 0.01 ug/kg to about 100 ug/kg.